Third Generation Sequencing Technologies for Microbiome analysis

May 31 2018

Third generation technology
Illumina Synthetic Long read technology
SMRT-Single Molecule Long Read technology
Nanopore Technologies
Hi-C Technology
Comparison of all technologies

Metagenomic sequencing (including 16s and whole genome sequencing), metatranscriptomics are common techniques applied to study microbial communities and their role in maintaining host health or in ecological stability. Popular sequencing technologies in meta-omics project include short read technologies such as Illumina, IonTorrent, 454 technologies that generate millions of short reads to give us a glimpse of microbial community. The bioinformatics community has developed tools to assemble these short sequences to longer sequences (to get a complete gene in one contiguous sequence), and predict the taxa/functions, reconstruct predicted genomes (called metagenome assembled genomes- MAG’s) with some confidence.

Third Generation technologies

Third generation sequencing technologies have had a drastic impact on eukaryotic studies, in improving reference genomes providing assemblies with near chromosome level completion (link here). Here is another blog post we already have on third-generation sequencing technologies, explaining each technology, their comparison, costs and some research papers that have applied them.

Stemming from the above blogpost, the focus here will be how these technologies offer current solutions to microbiome/metagenome studies (whole genome of course). In average a bacterial genome are about (~16kbp to 1.75Mbp), archaea (average of 2-5Mpb), viruses/phages (upto 250 kbp). Third generation sequencing technologies can sequence read lengths up to 100kbps. If you are working with microbial genomes this means you can get, near-complete to complete genome (no need to assemble – overcome assembly associated errors/biases), more confidence in gene annotation and more coverage of the genome (near-complete genomes can be put together with more confidence).

Illumina Synthetic Long read technology

This method is more or less the same as the current HiSeq Illumina technology with a difference in the manner of barcoding to get longer reads, here is a brief description on the technique. The sequencing prep kits are not distinct for microbial sequences specifically, remain the same for both eukaryotic genomes and microbial genomes. For more product information and specifications- go here.

Cost: ~$2500 per GB
Bioinformatics software: open source software are available
Research papers: Here is a pdf provided by Illumina with list of reviews and research studies using the long read technology

PacBio (SMRT-Single Molecule Long Read technology)

This method uses hairpin adaptors to attach the double strands and make circular DNA, and a polymerase sequence the entire circular strand over and over again, providing good coverage. Goto here for a brief description of how the technique works. They have three workflows, including full-length 16s sequencing, for metagenomics profiling and shotgun metagenome assemblies of near-complete genomes. Here is a link to PacBio’s workflows for microbial communities for all the three workflows. The kits offer up to ~10kbp read lengths, up to 100 fold coverage for 16s and ~50 per genome, for more information - click here or here.

The estimated cost of the process: $300-$400 per GB (source)
Bioinformatic software: Open source tools available: SMRT Tools or PacBio DevNet
Research papers - Here is a link from the PacBio website

Nanopore Technologies

Single-stranded sequence is passed through a nanopore that predicts the nucleotide-based on its size as it passes through a pore in a membrane. Goto here for a brief description of how the technique works. The real advantage of this technology is that the sequences are so small that can be connected to your laptop to a USB port allowing you to run the sequencer in lab or in the field. Here is a link to the Nanopore website with applications for microbiome studies.

Cost: $49.99 to $99.99 per Gb for MinIon - (source)
Bioinformatics software: Available on github: canu assembler, poretools, compiled list of tools.
Research papers: List from nanopore website

Hi-C Technology

Phase Genomics, have developed a ProxiMeta kit for metagenomic analysis. This method extracts the DNA sequence to keep the linkage between DNA molecules while sequencing to get 3D structure of the chromosome (link to blog). For microbiome/metagenome samples, the sequences from Hi-C technology are used as scaffolds to help with improving the Illumina contigs coverage of metagenome assembled genomes. Here is a description of how the kit works for metagenomes.

Cost: Sequencing prep kit is 2000$ for 2 samples. The sequencing itself can be run on Illumina sequencers.
Bioinformatics programs: ProxiMeta software (proprietary), there are open source programs available on github and other tools with active development.
Research papers: cow gut microbiome study, human microbiome studies

10X Technology

Applies Illumina short read technology but keeping track of the reads can be assembled to a larger template. For more information about the method- go here. Like the Illumina long read technology, the sequencing prep kits are not distinct for microbial sequences specifically.

Cost: -
Bioinformatics: Supernova for assembly, LongRanger analysis pipelines, another cloud-based assembler developed for microbiome sample Athena
Research papers: Human gut metagenome studies

Comparison of all technologies

See table